The overall goals of the projects under this investigator are to elucidate structure-function relationships in the surface proteins of enveloped viruses. Projects aim to dissect the mechanism of how viral envelope proteins mediate membrane fusion required for virus entry and how neutralizing and protective antibodies interfere with this entry process. Information gained from these studies will also be applied towards the rational design of novel viral inhibitors and vaccines. Vaccinia Entry Proteins. Research projects in this program aim to elucidate neutralizing antibody responses to vaccinia envelope proteins in the two major infectious forms of vaccinia virus, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). Neutralizing determinants in vaccinia immune globulin will also be analyzed. Information gained in these studies will aid the development and evaluation of novel vaccines to protect against smallpox. HIV envelope glycoprotein: structure, function, and vaccine potency. Enveloped viruses infect cells by fusing with cellular membranes. Fusion is mediated by viral envelope glycoproteins (Env) when they undergo conformational changes while binding target cells. HIV Env consists of a surface subunit (gp120) that binds receptor and a noncovalently-associated transmembrane subunit (gp41) that mediates membrane fusion. Gp120 binding to cellular receptors triggers conformational changes in the oligomeric Env complex that activates the fusion activity of gp41, allowing delivery of the viral capsid across the cell membrane. Our research projects aim to elucidate the entry mechanism and structure-function relationships of the HIV Env, in order to provide information for designing and evaluating novel HIV vaccines and inhibitors. We have been focusing on two heptad repeat regions (termed N- or C-heptad repeat according to proximity to the N-terminus of gp41) in the ectodomain of gp41 that self-assemble into a thermostable six-helix bundle. This structure (trimer of hairpins)consists of a parallel coiled-coil trimer (N heptad) with three C-heptad helices packed in the grooves of the coiled coil in an anti-parallel fashion. Peptides corresponding to the N- or C-heptad repeat regions of gp41 are potent inhibitors of HIV infection. They are believed to prevent fusion-inducing conformational changes in gp41 in a dominant-negative fashion, by binding to gp41 and interfering with formation of the gp41 six-helix bundle required for fusion. We are using biochemical, genetic, and immunological approaches to study HIV entry. We have successfully used the gp41 heptad-repeat peptides to trap Env undergoing conformational changes. These studies show that gp120 binding to receptors induces conformational changes that involve exposure of the two heptad-repeat regions in gp41 during HIV entry. Further studies are underway examining receptor/co-receptor requirements for Env triggering in various Envs. We have also undertaken extensive mutagenesis studies, examining the role of the six-helix bundle in membrane fusion. They studies reveal that the interhelical interactions between the N- and C-heptads are critically important for Env-mediated fusion. Further genetic studies are underway to refine such structure-function relationships in Env. Based on our findings that the gp41 heptad repeat are the targets of the inhibitory peptides and transiently exposed after receptor activation, we are generating antibodies to the heptad repeats in the gp41 fusion intermediates. This work involves rational design of novel immunogens/inhibitors and the generation and characterization of antibodies. Preliminary work indicates that antibodies can bind the fusion intermediates and block fusion under certain conditionsAdditional studies are underway to isolate high affinity antibodies to fusion-intermediate structures. Information gained in these studies will aid the development and evaluation of Env-based HIV vaccines. This project incorporates FY2002 projects 1Z01BK009006-07, 1Z01BK009009-06, 1Z01BK009010-04, and 1Z01BK009011-01.